Method for controlling streptococcus pneumoniae polysaccharide molecular weight using carbon dioxide

ABSTRACT

The present invention provides improved methods for producing a solution containing high molecular weight isolated  Streptococcus pneumoniae  capsular polysaccharides having phosphodiester linkages between saccharide repeat units. In certain methods, CO 2  is supplied to a fermentation culture of  Streptococcus pneumoniae  bacterial cells that produce capsular polysaccharide serotypes containing phosphodiester linkages between saccharide repeat units. Exemplary  Streptococcus pneumoniae  serotypes containing a phosphodiester linkage between saccharide repeat units include serotypes 6A, 6B, 19A, and 19F. Supplying CO 2  to the fermentation culture includes adding bicarbonate ions to the fermentation culture, adding carbonate ions to the fermentation culture, adding mixtures of bicarbonate and carbonate ions to the fermentation culture, and overlaying the fermentation culture with CO 2 .

This application claims benefit of priority to U.S. ProvisionalApplication No. 61/138,570, which was filed on 18 Dec. 2008, and whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for increasing the molecular weight ofisolated Streptococcus pneumoniae capsular polysaccharides having aphosphodiester linkage between saccharide repeat units.

BACKGROUND

In the preparation of multivalent conjugate pneumococcal vaccinesdirected to the prevention of invasive diseases caused by the organismStreptococcus pneumoniae (also known as pneumococcus), selectedStreptococcus pneumoniae serotypes are grown to supply polysaccharidesneeded to produce the vaccine. The cells are grown in fermentors withlysis induced at the end of the fermentation by addition of sodiumdeoxycholate or an alternate lysing agent. The lysate broth is thenharvested for downstream purification and the recovery of the capsularpolysaccharide which surrounds the bacterial cells. After conjugationwith a carrier protein, the polysaccharide is included in the finalvaccine product and confers immunity in the vaccine's target populationto the selected Streptococcus pneumoniae serotypes.

Polysaccharide size is a quality attribute assayed for in eachpreparation batch and must be appropriately controlled. Traditionalprocessing has involved using NaOH (sodium hydroxide) as a base titrantduring fermentation. The use of NaOH has the advantage of being able tolower the pH of the deoxycholate lysate without foaming to removeprotein and improve filtration. This material is subjected tocentrifugation followed by filtration to remove most of the solids downto a 0.45 μm nominal size. However, such traditional processing methodsresult in lower molecular weight polysaccharide (<450 kDa) for serotypeshaving a phosphodiester linkage between saccharide repeat units (e.g.,6A, 6B, 19A, and 19F).

Accordingly, improved methods for the recovery of high molecular weightcapsular polysaccharide from cellular Streptococcus pneumoniae lysates,in particular lysates containing Streptococcus pneumoniae serotype 6A,6B, 19A, or 19F polysaccharides, are needed.

BRIEF SUMMARY OF THE INVENTION

Improved methods for the recovery of high molecular weight capsularpolysaccharides from cellular Streptococcus pneumoniae lysatescontaining serotypes having a phosphodiester linkage between sacchariderepeat units are provided. In one method, CO₂ is supplied to afermentation culture of a Streptococcus pneumoniae serotype containing aphosphodiester linkage between saccharide repeat units. Accordingly, inone embodiment of the invention, the method includes the steps of: 1)preparing a fermentation culture of Streptococcus pneumoniae bacterialcells that produce capsular polysaccharides comprising a phosphodiesterlinkage between repeat units; 2) supplying CO₂ to the fermentationculture; 3) lysing the bacterial cells in the fermentation culture; and4) isolating Streptococcus pneumoniae capsular polysaccharides from thefermentation culture, where a solution containing high molecular weightisolated Streptococcus pneumoniae capsular polysaccharides containingphosphodiester linkages between repeat units is produced.

In a particular embodiment, fermentation cultures of Streptococcuspneumoniae bacterial cells that produce polysaccharide serotypes 19A,6A, 19F, 6B, and combinations thereof are prepared. In anotherparticular embodiment, supplying CO₂ to the fermentation cultureincludes adding bicarbonate ion (HCO₃ ⁻) to the fermentation culture,for example, adding NaHCO₃ (sodium bicarbonate) to the fermentationculture. In a further embodiment, supplying CO₂ to the fermentationculture includes adding carbonate ion (CO₃ ²⁻) to the fermentationculture, for example, adding Na₂CO₃ (sodium carbonate) to thefermentation culture. In another embodiment, supplying CO₂ to thefermentation culture includes a first addition of NaHCO₃ and a secondaddition of Na₂CO₃. In yet another embodiment, supplying CO₂ to thefermentation culture includes overlaying the fermentation culture withCO₂. In another embodiment, the molecular weight of the isolatedStreptococcus pneumoniae capsular polysaccharide is at least 700 kDa. Inanother embodiment, a solution containing high molecular weight isolatedStreptococcus pneumoniae capsular polysaccharides in which thepolysaccharides comprise phosphodiester linkages between repeat units isprovided, where the solution is produced by the method described above.

In another embodiment of the present invention, a method is provided forproducing a solution containing high molecular weight isolatedStreptococcus pneumoniae serotype 19A capsular polysaccharides. Themethod includes the steps of: 1) preparing a fermentation culture ofStreptococcus pneumoniae bacterial cells that produce serotype 19Acapsular polysaccharides; 2) supplying CO₂ to the fermentation culture;3) lysing the bacterial cells in the fermentation culture; and 4)isolating Streptococcus pneumoniae serotype 19A capsular polysaccharidesfrom the fermentation culture; whereby a solution containing highmolecular weight isolated Streptococcus pneumoniae serotype 19A capsularpolysaccharides is produced. In another embodiment, a solutioncontaining high molecular weight isolated Streptococcus pneumoniaeserotype 19A capsular polysaccharides is provided, where the solution isproduced by the method described above.

In another embodiment of the present invention, a method is provided forproducing a solution containing high molecular weight isolatedStreptococcus pneumoniae serotype 19F capsular polysaccharides. Themethod includes the steps of: 1) preparing a fermentation culture ofStreptococcus pneumoniae bacterial cells that produce serotype 19Fcapsular polysaccharides; 2) supplying CO₂ to the fermentation culture;3) lysing the bacterial cells in the fermentation culture; and 4)isolating Streptococcus pneumoniae serotype 19F capsular polysaccharidesfrom the fermentation culture; whereby a solution containing highmolecular weight isolated Streptococcus pneumoniae serotype 19F capsularpolysaccharides is produced. In another embodiment, a solutioncontaining high molecular weight isolated Streptococcus pneumoniaeserotype 19F capsular polysaccharides is provided, where the solution isproduced by the method described above.

In another embodiment of the present invention, a method is provided forproducing a solution containing high molecular weight isolatedStreptococcus pneumoniae serotype 6A capsular polysaccharides. Themethod includes the steps of: 1) preparing a fermentation culture ofStreptococcus pneumoniae bacterial cells that produce serotype 6Acapsular polysaccharides; 2) supplying CO₂ to the fermentation culture;3) lysing the bacterial cells in the fermentation culture; and 4)isolating Streptococcus pneumoniae serotype 6A capsular polysaccharidesfrom the fermentation culture; whereby a solution containing highmolecular weight isolated Streptococcus pneumoniae serotype 6A capsularpolysaccharides is produced. In another embodiment, a solutioncontaining high molecular weight isolated Streptococcus pneumoniaeserotype 6A capsular polysaccharides is provided, where the solution isproduced by the method described above.

In another embodiment of the present invention, a method is provided forproducing a solution containing high molecular weight isolatedStreptococcus pneumoniae serotype 6B capsular polysaccharides. Themethod includes the steps of: 1) preparing a fermentation culture ofStreptococcus pneumoniae bacterial cells that produce serotype 6Bcapsular polysaccharides; 2) supplying CO₂ to the fermentation culture;3) lysing the bacterial cells in the fermentation culture; and 4)isolating Streptococcus pneumoniae serotype 6B capsular polysaccharidesfrom the fermentation culture; whereby a solution containing highmolecular weight isolated Streptococcus pneumoniae serotype 6B capsularpolysaccharides is produced. In another embodiment, a solutioncontaining high molecular weight isolated Streptococcus pneumoniaeserotype 6B capsular polysaccharides is provided, where the solution isproduced by the method described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the optical density (OD), base and glucose levels duringthe fermentation phase with Na₂CO₃ as titration base from laboratorystudies at 3 L scale. Base in grams is divided by 10 for plottingpurposes.

FIG. 2 shows the optical density (OD), base and glucose levels duringthe fermentation phase with NaOH as titration base from laboratorystudies at 3 L scale. Base in grams is divided by 10 for plottingpurposes.

FIG. 3 shows total protein and polysaccharide results at different pHadjustments for alternate base feeds of Na₂CO₃ or NaOH.

DETAILED DESCRIPTION OF THE INVENTION

Streptococcus pneumoniae are Gram-positive, lancet shaped cocci that areusually seen in pairs (diplococci), but also in short chains or assingle cells. They grow readily on blood agar plates with glisteningcolonies and display alpha hemolysis unless grown anaerobically wherethey show beta hemolysis. The cells of most pneumococcal serotypes havea capsule which is a polysaccharide coating surrounding each cell. Thiscapsule is a determinant of virulence in humans, as it interferes withphagocytosis by preventing antibodies from attaching to the bacterialcells. Currently there are more than 90 known pneumococcal capsularserotypes identified, with the 23 most common serotypes accounting forapproximately 90% of invasive disease worldwide.

As a vaccine, the pneumococcal polysaccharide coat can confer areasonable degree of immunity to Streptococcus pneumoniae in individualswith developed or unimpaired immune systems, but a conjugated proteinwith polysaccharide allows for an immune response in infants and elderlywho are also most at risk for pneumococcal infections. The pneumococcalcells are grown in fermentors with lysis induced at the end of thefermentation. The lysate broth is then harvested for downstreampurification and the recovery of the capsular polysaccharides.

Polysaccharide size is a quality attribute assayed for in eachpreparation batch and must be appropriately controlled. The molecularweight for serotypes having a phosphodiester linkage between sacchariderepeat units (e.g., 6A, 6B, 19A, and 19F) is affected by parameters ofthe fermentation process. The methods of the present invention allow forthe recovery of high molecular weight capsular polysaccharides fromcellular Streptococcus pneumoniae lysates containing serotypes having aphosphodiester linkage between saccharide repeat units, such as serotype6A, serotype 6B, serotype 19A, serotype 19F, and combinations thereof.

In the development of the present methods, the concentration of HySoyand choice of base titrant were modified in an attempt to modify finalpolysaccharide yields and molecular weights. Four fermentation schemeswere tested. The first used a baseline NaOH process with 28 g/L HySoy.The second used 20% sodium carbonate as the base titrant and 20 g/LHySoy. The third combined advantages of the first two approaches byintroducing carbonate through the batching of sodium bicarbonate andusing a mixed NaOH/carbonate base titrant. The fourth approach usedcarbonate as the base titrant with a 10 mM bicarbonate addition tobolster growth.

Using NaOH as the base titrant during fermentation had the advantage ofbeing able to lower the deoxycholate lysate to pH 5.0 without foaming toremove protein and improve filtration, but resulted in lower molecularweight polysaccharide (<450 kDa). Na₂CO₃ provided higher molecularweight but had foaming issues if the pH of the deoxycholate lysate waslowered. At a higher pH hold step of 6.6, the fermentations using Na₂CO₃formed a gel-like material, with subsequent filtration problems.Minimizing the amount of Na₂CO₃ by using a blend of NaOH and Na₂CO₃ as apH titrant provided the molecular weight size benefits of Na₂CO₃ whileeliminating foaming and filtration problems due to the sudden release oflarge amounts of CO₂. The use of 20% Na₂CO₃ (w/v) as the base titrantwith a 10 mM NaHCO₃ addition to bolster growth (fourth approach)produced consistent, high molecular weight polysaccharides at highyield.

The present invention thus provides improved methods for the recovery ofhigh molecular weight capsular polysaccharides from cellularStreptococcus pneumoniae lysates containing serotypes having aphosphodiester linkage between saccharide repeat units. In one method,CO₂ is supplied to a fermentation culture of a Streptococcus pneumoniaeserotype containing a phosphodiester linkage between saccharide repeatunits. Exemplary Streptococcus pneumoniae serotypes containing aphosphodiester linkage between saccharide repeat units include serotypes6A, 6B, 19A, and 19F. Accordingly, in one embodiment of the invention, amethod for producing a solution containing high molecular weightisolated Streptococcus pneumoniae capsular polysaccharides that comprisephosphodiester linkages between repeat units is provided, which includesthe steps of: 1) preparing a fermentation culture of Streptococcuspneumoniae bacterial cells that produce capsular polysaccharidescomprising a phosphodiester linkage between repeat units; 2) supplyingCO₂ to the fermentation culture; 3) lysing the bacterial cells in thefermentation culture; and 4) isolating Streptococcus pneumoniae capsularpolysaccharides from the fermentation culture; whereby a solutioncontaining high molecular weight isolated Streptococcus pneumoniaecapsular polysaccharides with phosphodiester linkages between repeatunits is produced. In another embodiment, the present invention relatesto a solution containing high molecular weight isolated Streptococcuspneumoniae capsular polysaccharides with phosphodiester linkages betweenrepeat units, where the solution is produced by the method describedabove.

The methods of the invention produce high molecular weight Streptococcuspneumoniae capsular polysaccharides that comprise phosphodiesterlinkages between repeat units (for example, serotypes 6A, 6B, 19A, and19F). As used herein, “high molecular weight” refers to molecularweights that are at least about 480 kDa, about 490 kDa, about 500 kDa,about 510 kDa, about 520 kDa, about 525 kDa, about 550 kDa, about 575kDa, about 600 kDa, about 625 kDa, about 650 kDa, about 675 kDa, about700 kDa, about 725 kDa, about 750 kDa, about 775 kDa, about 800 kDa,about 825 kDa, about 850 kDa, about 875 kDa, about 900 kDa, about 925kDa, about 950 kDa, about 975 kDa, or about 1000 kDa.

In certain methods, supplying CO₂ to the fermentation culture includesadding bicarbonate ion (HCO₃ ⁻) to the fermentation culture, forexample, adding NaHCO₃ to the fermentation culture. NaHCO₃ additions of5-50 mM can be used, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35mM, 40 mM, 45 mM, or 50 mM. In other methods, supplying CO₂ to thefermentation culture includes adding carbonate ion (CO₃ ²⁻) to thefermentation culture, for example, adding Na₂CO₃ to the fermentationculture. Na₂CO₃ additions of 0.1 M-2.0 M can be used, such as 0.1 M, 0.2M, 0.4 M, 0.6 M, 0.7 M, 0.9 M, 1.0 M, 1.5 M, 1.8 M, or 2.0 M. Aweight/volume (w/v) equivalent can also be used, such as 5% (w/v)Na₂CO₃, 10% (w/v) Na₂CO₃ or 20% (w/v) Na₂CO₃. In yet other methods,supplying CO₂ to the fermentation culture includes a first addition ofNaHCO₃ and a second addition of Na₂CO₃ to the fermentation culture. Infurther methods, supplying CO₂ to the fermentation culture includesoverlaying the fermentation culture with CO₂. CO₂ overlays of 5%-100%can be used, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Within the methods of the present invention, the bacterial cells may belysed using any lytic agent. A “lytic agent” is any agent that aids incell wall breakdown and release of autolysin which causes cellular lysisincluding, for example, detergents. As used herein, the term “detergent”refers to any anionic or cationic detergent capable of inducing lysis ofbacterial cells. Representative examples of such detergents for usewithin the methods of the present invention include deoxycholate sodium(DOC), N-lauryl sarcosine (NLS), chenodeoxycholic acid sodium, andsaponins.

In one embodiment of the present invention, the lytic agent used forlysing bacterial cells is DOC. DOC is the sodium salt of the bile aciddeoxycholic acid, which is commonly derived from biological sources suchas cows or oxen. DOC activates the LytA protein, which is an autolysinthat is involved in cell wall growth and division in Streptococcuspneumoniae. The LytA protein has choline binding domains in itsC-terminal portion, and mutations of the lytA gene are known to produceLytA mutants that are resistant to lysis with DOC.

Although there is no evidence that the use of DOC during Streptococcuspneumoniae polysaccharide purification poses a health risk, the use ofsuch biologically derived reagents could raise potential regulatoryconcerns. Accordingly, in one embodiment of the present invention, thelytic agent used for lysing bacterial cells is a non-animal derivedlytic agent. Non-animal derived lytic agents for use within the methodsof the present invention include agents from non-animal sources withmodes of action similar to that of DOC (i.e., that affect LytA functionand result in lysis of Streptococcus pneumoniae cells). Such non-animalderived lytic agents include, but are not limited to, analogs of DOC,surfactants, detergents, and structural analogs of choline, and may bedetermined using procedures as described in the Experimental sectionherein below. In one embodiment, the non-animal derived lytic agent isselected from the group consisting of decanesulfonic acid,tert-octylphenoxy poly(oxyethylene)ethanols (e.g. Igepal® CA-630, CAS #:9002-93-1, available from Sigma Aldrich, St. Louis, Mo.), octylphenolethylene oxide condensates (e.g. Triton® X-100, available from SigmaAldrich, St. Louis, Mo.), N-lauryl sarcosine sodium (NLS), lauryliminodipropionate, sodium dodecyl sulfate, chenodeoxycholate,hyodeoxycholate, glycodeoxycholate, taurodeoxycholate,taurochenodeoxycholate, and cholate. In another embodiment, thenon-animal derived lytic agent is NLS.

Within the methods of the present invention, Streptococcus pneumoniaecapsular polysaccharides are isolated using standard techniques known tothose skilled in the art. For example, following fermentation ofbacterial cells that produce Streptococcus pneumoniae capsularpolysaccharides, the bacterial cells are lysed to produce a cell lysate.The capsular polysaccharides may then be isolated from the cell lysateusing purification techniques known in the art, including the use ofcentrifugation, precipitation, ultra-filtration, and columnchromatography (See, for example, U.S. Patent App. Pub. Nos.20060228380, 20060228381, 20070184071, 20070184072, 20070231340, and20080102498).

The process changes described above allow for the recovery of highmolecular weight capsular polysaccharides from cellular Streptococcuspneumoniae lysates containing serotypes having a phosphodiester linkagebetween saccharide repeat units, such as serotype 6A, serotype 6B,serotype 19A, serotype 19F, and combinations thereof. This is a robustimprovement of the fermentation/recovery process that can greatlyenhance the production of pneumococcal polysaccharides.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Selected Streptococcus pneumoniae serotypes were grown to supplypolysaccharides needed to produce vaccines for active immunizationagainst invasive disease caused by Streptococcus pneumoniae due tocapsular serotypes included in the vaccine. The cells were grown infermentors with lysis induced at the end of the fermentation. The lysatebroth was then harvested for downstream purification and the recovery ofthe capsular polysaccharides. Because polysaccharide size is a qualityattribute assayed for in each preparation batch, polysaccharide sizemust be appropriately controlled. The molecular weight for serotypeshaving a phosphodiester linkage between saccharide repeat units (e.g.,6A, 6B, 19A, and 19F) was found to be affected by parameters of thefermentation process. The following example describes studies relatingto the supply of CO₂ during fermentation of Streptococcus pneumoniaeserotypes having a phosphodiester linkage between repeat units toimprove polysaccharide molecular weight.

Example 1 Carbon Dioxide Supply Effect on Polysaccharide MolecularWeight Fermentation

Laboratory runs were performed in 3 L Braun Biostat B fermentors (B.Braun Biotech, Allentown, Pa.). They were filled with 1.8 L of HYSmedium (20 g/L HySoy, 2.5 g/L NaCl, 0.5 g/L KH₂PO₄, 0.013 g/LCaCl₂.2H₂O, 0.15 g/L L-Cysteine HCl). The fermentors were thenautoclaved for 60 minutes at 121° C. After cooling, either 40 or 60 mL/Lof a 50% Glucose+1% Magnesium Sulfate (w/v) (DMS) solution was added toeach unit. If required, sodium bicarbonate was added prior toinoculation.

Two 2 L seed bottles containing 1 L of HYS media were inoculated withType 19A or Type 6A frozen seed stocks and incubated at 36° C. withoutshaking for approximately 6-8 hours. Inoculation of the fermentors wasperformed with a volume of 100 mL (˜5.2% v/v) aliquoted from a bottlewith an OD₆₀₀ between 0.3-0.9 and pH between 4.75-5.60. The fermentationtemperature and pH were controlled at the desired setpoints. Thestandard conditions of 36° C., 1 L/min air overlay, pH controlled to 7and agitation of 75 rpm were used. Two impellers were placed at the lowand middle positions on the agitator shaft. A bottle containing theappropriate base titrant (3 N NaOH, 3 N NaOH blended with variousconcentrations of NaHCO₃, 3 N NaOH blended with various concentrationsof Na₂CO₃ and NaHCO₃, and 20% Na₂CO₃) was hooked up for automatic pHcontrol. The fermentors were sampled at various time points for externalpH, OD₆₀₀, glucose, polysaccharide, and protein. The runs wereterminated when the glucose concentration was near depletion, or noincrease in OD over time was noted.

Optical Density (OD₆₀₀) Measurement

The cellular density of the fermentation broth was determined by readingthe absorbance of the samples at 600 nm using a Shimadzu (Columbia, Md.)UV-1601 (2 nm bandwidth) or Spectronics (Westbury, N.Y.) Genesys 5spectrophotometer (5 nm bandwidth). The unit was blanked with the HYSmedium diluted with de-ionized (DI) water to match the dilution requiredof the sample. The sample was diluted to keep the absorbance below areading of 0.4, which is well within the linear range of thespectrophotometer.

Glucose Concentration

Glucose levels were determined by centrifuging out the cells and usingthe supernatant straight or 3× diluted with DI water. The samples wereanalyzed on a Nova Biomedical (Waltham, Mass.) BioProfile 400.

Polysaccharide Analysis

Samples were taken at the final fermentation reading and treated with12% sodium deoxycholate (DOC) to a concentration of 0.13% (w/v) andgently agitated. They were held between 8-24 hours at 5° C. then pHadjusted to 5.0 with 50% acetic acid to precipitate out most of the DOCand protein. After another hold interval of 12-24 hours at 5° C., thesamples were centrifuged (14000 rpm, Sorvall (Thermo Fisher Scientific,Waltham, Mass.) SS34 rotor, 10 min at 15° C.). The pH of the supernatantwas adjusted to 6.0. The supernatant was then filtered through 0.45 μmPall (East Hills, N.Y.) HT Tuffryn Membrane syringe filters (low proteinbinding). The filtered product was analyzed by high-performance sizeexclusion chromatography (HPLC-SEC) using standard methodology wellknown in the art (see, e.g., Aguilar, M. “HPLC of Peptides and Proteins:Methods and Protocols” Totowa, N.J.: Humana Press (2004)).

Protein Analysis

Protein levels were analyzed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) methods well known in the art (see, e.g.,Walker, J. M. “The Protein Protocols Handbook” Totowa, N.J.: HumanaPress (2002)). The filtered cell lysate (supernatant) as prepared abovewas aliquoted into microfuge tubes at 65 μL/tube. Additions of reducingagent (10 μL dithiothreitol (DTT)) and NuPAGE® (Invitrogen, Carlsbad,Calif.) 4× lithium dodecyl sulfate (LDS) sample buffer (25 μL) were madeto each sample. The samples were vortexed and heated for 10 minutesprior to 10 μL/lane loading on NuPAGE® 4-12% Bis-Tris 12 well gels. Thegels were run in NuPAGE®MES-SDS buffer at 150 V limiting forapproximately 60 minutes and subsequently stained using the Zoionstaining protocol (Zoion Biotech, Worcester, Mass.). Sample analysis wasperformed using an UVP Imager (UVP Inc., Upland, Calif.) with LabWorks™(UVP Inc.) V.3 software to obtain approximate concentrations of specificprotein bands of interest. Bovine Serum Albumin (BSA) Fraction V wasused to develop a protein standard curve to calculate the approximateprotein values of the samples (in lysed cell broth).

Molecular Weight Analysis

Fermentation samples of 1-2 liters were treated with 12% sodium DOC to aconcentration of 0.13% (w/v) with agitation at 200 rpm. Samples wereheld between 8-24 hours at either 5° C. or 20° C. The samples were thenpH adjusted to 5.0 or 6.6 with 50% acetic acid to precipitate out mostof the DOC and protein. After another hold interval of 12-24 hours at 5°C., the samples were centrifuged (11000 rpm, Sorvall (Thermo FisherScientific, Waltham, Mass.) SLA-3000 rotor, 15 min at 10° C.). Thesupernatant samples were then pH adjusted to 6.0 with 3 N NaOH, andfiltered using 0.45 μm Millipore (Billerica, Mass.) MP60 filters. Thesamples were then subjected to a modified purification processconsisting of 100K molecular weight cut-off (MWCO) diafiltration (5×concentration followed by 7.5× diafiltration with DI water), 0.1% HBprecipitation, and carbon filtration. The purified material was thensubjected to Multi Angle Laser Light Scattering (MALLS) analysis.

Fermentation Process Study

Based on previous studies, the fermentation process was optimized byswitching from Na₂CO₃ to NaOH as the base titrant. Use of NaOH allowedthe recovery pH to be lowered to 5.0 resulting in significant proteinprecipitation. Na₂CO₃ will release CO₂ at low pH (<6.6) creating foamformation. The impact of base titrant on Type 19A polysaccharide andprotein levels was examined. Two 3 L fermentors were set up with onefermentor serving as the original process control, using 20% Na₂CO₃solution (w/v) as the base feed. The other fermentor used 3 N NaOH asthe base feed. During the recovery phase, cells were lysed in thefermentor with DOC (final concentration 0.13% (w/v)) with the fermentorheld at 36° C. for 30 minutes. Following this step, the lysate was heldovernight with agitation at ambient temperature (22° C.). After thelysate hold, the lysate was pH titrated through a range from unadjustedto 4.5 with samples pulled at various pH setpoints. These samples wereheld overnight at ambient temperature prior to being processed andanalyzed for polysaccharide and protein concentrations. The OD, base andglucose levels during the fermentation phase are shown in FIG. 1 andFIG. 2. The major difference was a higher final OD for the carbonaterun.

The effect of post DOC lysate pH adjustment on total protein levels wasalso examined, and is shown in FIG. 3. The lower pH levels reduced theprotein load in cell free broth for both the NaOH run and the Na₂CO₃run. The lower pH (<6.6) had no negative impact on the polysaccharideyield. The fermentation analysis results served as an indication thatthe NaOH base feed was an acceptable alternative to the process usingthe Na₂CO₃ base feed during fermentation, but produced lower yields thanwhat was obtained with the Na₂CO₃ feed.

Effect of Base Titrant on 19A and 6A Molecular Weight

A set of fermentations at the 3 L scale were performed to determine ifthe base titrant, HySoy concentration and pH hold step affected serotype19A molecular weight. The molecular weight determination was performedusing MALLS assay following the modified purification process. Resultsare shown in Table 1. For serotype 6A, only the base titrant wasevaluated. Results are shown in Table 2.

TABLE 1 Impact of base titrant on 19A molecular weight (L29331-94) MALLSRun No. pH/Temp HySoy pH Hold Base (kDa) D 7.0/36° C. 28 g/L 5.0 3 NNaOH 340 E 7.0/36° C. 20 g/L 5.0 3 N NaOH 350 F 7.0/36° C. 20 g/L 5.020% Na₂CO₃ 713 H 7.0/36° C. 20 g/L 6.6 20% Na₂CO₃ 713

TABLE 2 Impact of base titrant on 6A molecular weight MALLS Run No. Base(kDa) Lab 1 3 N NaOH 662 Lab 2 20% Na₂CO₃ 1189 Pilot 1 3 N NaOH 500Pilot 2 20% Na₂CO₃ 950

Effect of Bicarbonate and Mixed Base pH Titrant

In the first study (Runs L29331-122 and -139), varying levels of initialsodium bicarbonate and base blends of sodium hydroxide and sodiumcarbonate were used in conjunction with a pH 5.0 hold step after the DOChold step. The initial bicarbonate additions ranged from 10-50 mM andthe sodium carbonate added to 3N sodium hydroxide for the base titrantranged from 0.2-1.8 M. One run contained 50 mM initial bicarbonate andused NaOH as a base titrant. The carbonate levels at the end of thesefermentations ranged from 14-111 mM. Serotype 19A molecular weightsranged from 520 to 713 kDa. Run parameters and results are shown inTable 3.

TABLE 3 Na₂CO₃ vs. mixed base as pH titrant NaHCO₃ Base MALLS PS YieldRun No. (mM) Na₂CO₃ NaOH (kDa) (mg/mL) Part I E 0 20% 0 759 0.836L29331-122 F 10 0.2 M 3 N 520 0.308 20 g/L HySoy G 10 0.4 M 3 N 6480.538 H 10 0.9 M 3 N 563 0.334 Part II C 20 0.9 M 3 N 662 1.027 L29331-D 20 1.8 M 3 N 611 0.903 139 G 50 0.9 M 3 N 713 0.924 28 g/L H 50   0 M3 N 713 1.051 HySoy

A second study (L29331-159 and -185) used initial bicarbonate additionsof 15-30 mM and base blends using 0.4-1.0 M Na₂CO₃. The carbonate levelsat the end of fermentation ranged from 24-62 mM. Serotype 19A molecularweights ranged from 502 to 763 kDa. Run parameters and results are shownin Table 4.

TABLE 4 NaHCO₃ with mixed base as pH titrant Run NaHCO₃ Na₂CO₃/ MALLS PSYield No. HySoy/DMS (mM) NaOH (kDa) (mg/mL) G2 28 g/L/60 mL/L 15 1.0 M/3N 657 0.853 H2 28 g/L/60 mL/L 15 0.4 M/3 N 605 0.755 C 20 g/L/60 mL/L 200.4 M/3 N 571 0.386 E 20 g/L/60 mL/L 20 1.0 M/3 N 763 0.439 F 20 g/L/60mL/L 25 0.7 M/3 N 462 0.382 G 20 g/L/60 mL/L 30 0.4 M/3 N 502 0.355 H 20g/L/60 mL/L 30 1.0 M/3 N 594 0.415

Comparison of Mixed and Pure Carbonate Titration Base FermentationProcesses

An experiment was performed to compare the base blend process (0.7 MNa₂CO₃/3 N NaOH) to the carbonate titrant process (20% Na₂CO₃ solution,w/v). Results (Table 5) confirmed that the molecular weight from thecarbonate titrant process was higher and more consistent (778, 781 kDa)than the molecular weight from the base blend process (561-671 kDa).Polysaccharide yield was also higher with the Na₂CO₃ process.

TABLE 5 Run L29399-1 Na₂CO₃ vs. mixed base NaHCO₃ Base MW PS Yield RunNo. (mM) Na₂CO₃ NaOH (kDa) (mg/mL) C 25 0.7 M 3 N 565 1.106 D 25 0.7 M 3N 561 0.908 E 25 0.7 M 3 N 612 0.894 G 25 0.7 M 3 N 671 0.873 F 0 20% 0778 1.282 H 0 20% 0 781 1.249

Pilot Scale Runs

Several serotype 19A pilot scale (100 L) runs with various base titrantswere performed. The molecular weight determination was performed usingMALLS assay following a complete purification process and is reportedfrom the final purified batch. Results are shown in Table 6.

TABLE 6 Impact of base titrant on 19A molecular weight at pilot scaleFBC Fermentation Purification MALLS Batch Titration Base Batch (kDa)RRP19A-0008 3 N NaOH L26563-10 390 RRP19A-0009 3 N NaOH L26563-11 380IPPPN19A-005 3 N NaOH/0.6 M Na₂CO₃ L26260-37 492 IPPPN19A-006 3 NNaOH/0.6 M Na₂CO₃ L26260-38 480 IPPPN19A-007 3 N NaOH/0.6 M Na₂CO₃L26260-39 490 IPPPN19A-014 20% Na₂CO₃ L26260-49 580 IPPPN19A-016 20%Na₂CO₃ L26260-50 559 IPPPN19A-017 20% Na₂CO₃ L26260-51 599

Effect of Base Titrant and Overlay on 19A Molecular Weight

A set of fermentations at the 3 L scale were performed to determine ifthe base titrant and atmospheric overlay affected the molecular weight.The molecular weight determination was performed using MALLS assayfollowing the modified purification process. Results are shown in Table7.

TABLE 7 Impact of base titrant and overlay on 19A molecular weight MALLSRun No. Base Overlay (kDa) Control 3 N NaOH Air 350 C 0.7 M Na₂CO₃ Air855 D 1.5 M Na₂CO₃/ Air 710 1.5 N NaOH E 3 N NaOH 100% CO₂  634 F 3 NNaOH 50% CO₂ 646 G 3 N NaOH 20% CO₂ 567 H 3 N NaOH 10% CO₂ 547

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. A method for producing a solution containing high molecular weightisolated Streptococcus pneumoniae capsular polysaccharides wherein saidpolysaccharides comprise phosphodiester linkages between repeat units,the method comprising: a) preparing a fermentation culture ofStreptococcus pneumoniae bacterial cells that produce capsularpolysaccharides comprising a phosphodiester linkage between repeatunits; b) supplying CO₂ to said fermentation culture; c) lysing thebacterial cells in said fermentation culture; and d) isolatingStreptococcus pneumoniae capsular polysaccharides from said fermentationculture; whereby a solution containing high molecular weight isolatedStreptococcus pneumoniae capsular polysaccharides wherein saidpolysaccharides comprise phosphodiester linkages between repeat units isproduced.
 2. The method of claim 1, wherein said Streptococcuspneumoniae capsular polysaccharides are serotype 19A.
 3. The method ofclaim 1, wherein said Streptococcus pneumoniae capsular polysaccharidesare serotype 6A.
 4. The method of claim 1, wherein said Streptococcuspneumoniae capsular polysaccharides are serotype 19F.
 5. The method ofclaim 1, wherein said Streptococcus pneumoniae capsular polysaccharidesare serotype 6B.
 6. The method of claim 1, wherein supplying CO₂ to saidfermentation culture comprises adding bicarbonate ion (HCO₃ ⁻) to thefermentation culture.
 7. The method of claim 6, wherein adding HCO₃ ⁻ tothe fermentation culture comprises adding NaHCO₃.
 8. The method of claim1, wherein supplying CO₂ to said fermentation culture comprises addingcarbonate ion (CO₃ ²⁻) to the fermentation culture.
 9. The method ofclaim 8, wherein adding CO₃ ²⁻ to the fermentation culture comprisesadding Na₂CO₃.
 10. The method of claim 9, further wherein the pH of saidfermentation culture is between 6.0 and 6.6.
 11. The method of claim 1,wherein supplying CO₂ to said fermentation culture comprises a firstaddition of NaHCO₃ and a second addition of Na₂CO₃.
 12. The method ofclaim 1, wherein supplying CO₂ to said fermentation culture comprisesoverlaying the fermentation culture with CO₂.
 13. The method of claim 1,wherein lysing the Streptococcus pneumoniae in said fermentation culturecomprises adding sodium deoxycholate to said fermentation culture. 14.The method of claim 1, wherein the molecular weight of said isolatedStreptococcus pneumoniae capsular polysaccharide is at least 480 kDa.15. A solution containing high molecular weight isolated Streptococcuspneumoniae capsular polysaccharides wherein said polysaccharidescomprise phosphodiester linkages between repeat units, wherein saidsolution is produced by the method of claim 1.